At the Battle of Chancellorsville in May 1863, Barksdale's Brigade was one of the few units in James Longstreet's Corps that was present at the battle; most of the corps was detached for duty in Suffolk, Virginia. Once again, Barksdale's brigade defended the heights above Fredericksburg, this time against his previous adversary, Sedgwick, whose VI Corps was over ten times the size of his brigade. Sedgwick's assault was successful and Barksdale pulled back after delaying the Union force, but he was able to rally his brigade and retake the lost ground the next day.

At the Battle of Gettysburg, Barksdale's Brigade arrived with McLaws's Division after the first day of battle, July 1, 1863. The plan from General Robert E. Lee was for Longstreet's Corps to maneuver into position and attack northeast, up the Emmitsburg Road, to roll up the Union left flank. Barksdale's sector of the attack placed him directly at the tip of the salient in the Union line anchored at the Peach Orchard, defended by the Union III Corps. At about 5:30 p.m., Barksdale's Brigade burst from the woods and started an irresistible assault, which has been described as one of the most breathtaking spectacles of the Civil War. A Union colonel was quoted as saying, "It was the grandest charge that was ever made by mortal man."[2] Although he ordered his subordinate commanders to walk during the charge, Barksdale himself rode on horseback "in front, leading the way, hat off, his wispy hair shining so that it reminded [a Confederate staff officer] of 'the white plume of Navarre'."[3]

The Confederates smashed the brigade manning the Peach Orchard line, wounding and capturing the Union brigade commander himself. Some of Barksdale's regiments turned to the north and shattered Maj. Gen. Andrew A. Humphreys's division. Others of his regiments went straight ahead. By the time his men had gone as far as Plum Run, a mile into the assault, they were counterattacked by a brigade under Colonel George L. Willard. Barksdale was wounded in his left knee, followed by a cannonball to his left foot, and finally was hit by another bullet to his chest, knocking him off his horse. He told his aide, W.R. Boyd, "I am killed! Tell my wife and children that I died fighting at my post."[4] His troops were forced to leave him for dead on the field and he died the next morning in a Union field hospital (the Joseph Hummelbaugh farmhouse).

Barksdale is also featured in the 2011 History Channel film Gettysburg.

The streets in the Potomac Crossing subdivision in Leesburg, Virginia, are named (in part) after the regimental commanders of the Battle of Ball's Bluff (October 21, 1861). Barksdale Drive was named for Col. Barksdale and is the primary east-west conduit in the development, running just short of a mile to either end of the neighborhood.